*Target (protein/gene name): 6-phosphogluconate dehydrogenase *NCBI Gene # or RefSeq#:5475141 *Protein ID (NP or XP #) or Wolbachia#: PVX_117745 (PlasmoDB) *Organism (including strain): Plasmodium vivax Etiologic Risk Group (see link below): */Disease Information (sort of like the Intro to your Mini Research Write up): Plasmodium vivax is a protozoal parasite that is the main source of malaria. P. vivax is one of six species of malaria carriers that infect humans.
Symptoms include fever, chills, headache, muscular aching and weakness, vomiting, cough, diarrhoea and abdominal pain. According to DNDi (Drugs for Neglected Diseases initiative), malaria kills about one child every 30 seconds. Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=268632 Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) Essentiality of this protein: Gene/Ortholog: mtu1874 (OG4_10600); Phenotype: non-essential; Source study: nmpdr Gene/Ortholog: mtu1142 (OG4_10600); Phenotype: essential; Source study: nmpdr Gene/Ortholog: eco1968 (OG4_10600); Phenotype: undefined; Source study: blattner Gene/Ortholog: eco1968 (OG4_10600); Phenotype: non-essential; Source study: gerdes Gene/Ortholog: eco1968 (OG4_10600); Phenotype: non-essential; Source study: keio Gene/Ortholog: eco1968 (OG4_10600); Phenotype: non-essential; Source study: shigen Gene/Ortholog: cel16317 (OG4_10600); Phenotype: Larval/Adult Lethal/Arrest; Source study: neb Gene/Ortholog: cel16317 (OG4_10600); Phenotype: Embryonic Lethal/Arrest; Source study: neb Gene/Ortholog: cel16317 (OG4_10600); Phenotype: Embryonic Lethal/Arrest; Source study: wormbase Gene/Ortholog: cel16317 (OG4_10600); Phenotype: Larval/Adult Lethal/Arrest; Source study: wormbase Gene/Ortholog: Tb09.211.3180 (OG4_10600); Phenotype: significant loss of fitness in bloodstream forms (3 days); Source study: alsford Gene/Ortholog: Tb09.211.3180 (OG4_10600); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford Gene/Ortholog: Tb09.211.3180 (OG4_10600); Phenotype: significant loss of fitness in procyclic forms; Source study: alsford Gene/Ortholog: Tb09.211.3180 (OG4_10600); Phenotype: significant loss of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Complex of proteins?: No, but the active site may be in chain A or B Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.8
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: Homology model: PDB 2W8Z w/ lig
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors: Expression Information (has it been expressed in bacterial cells): expressed in E. coli bacterial cells Purification Method: Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MTGTCDIGLIGLAVMGQNLSLNIASNGFTIGVYNRTYERTEDTLKKAKEGNLPIQGYETL EQLINNLKKPRKIILLIKAGPAVDETIKNILKHFEEGDIIIDGGNEWYLNTERRITLCEE HKVEYLAMGVSGGEAGARYGCSFMPGGSKYAYDTIKDILEKCSAKVGTSPCVTYIGPRSS GNYVKMVHNGIEYGDMQLISESYLLMKNILNYNNEKLSEVFKKWNEGILNSYLIEITYKI LGKKDELTDNHLVDMILDIAGAKGTGKWTMLEAIERGIPCPTMCAALDARNISAYKQLRV KADSHFSNDMKVNKVEGENLINFEDDLLNALYCCKIISYTQGLFLLKQVSEEMKWNLNLG EISRIWRGGCIIRAVFLDRITNAYKKNEKLDLLFLDEDFAQEMKNKLPSLRKVVQVATKS SIPIPAFSASLAYFQMVTSQNLPLNLVQAQRDYFGSHTYKRVDRDGDFHTIWE *length of your protein in Amino Acids: 473 aa Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 53372.4 Molar Extinction coefficient of your protein at 280 nm wavelength: 66405 assuming all pairs of Cys residues form cystines
2 strong possible transmembrane regions according to TMPred Expasy
*CDS Gene Sequence (paste as text only): *GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): TACTTCCAATCCATGACCGGCACTTGTGATATCGGTCTCATTGGTCTGGCGGTTATGGGTCAGAACCTGTCC CTGAACATCGCCTCTAACGGTTTTACCATCGGTGTTTACAACCGTACCTACGAGCGTACG GAAGATACCCTGAAGAAAGCGAAAGAGGGTAACCTGCCGATCCAAGGTTATGAAACCCTC GAGCAGCTGATCAACAACCTCAAGAAACCGCGTAAAATCATTCTGCTCATCAAAGCGGGT CCGGCGGTTGACGAAACCATTAAGAACATCCTGAAGCACTTTGAGGAAGGCGACATCATC ATCGACGGTGGTAACGAATGGTACCTCAATACCGAACGTCGTATCACTCTGTGCGAAGAG CACAAAGTAGAATACCTGGCGATGGGTGTTTCTGGTGGCGAAGCAGGTGCGCGCTATGGC TGTTCTTTCATGCCGGGTGGCTCCAAATATGCGTATGACACGATCAAAGACATCCTCGAG AAGTGCTCTGCGAAAGTTGGCACGAGCCCTTGCGTAACTTACATCGGTCCTCGTAGCTCT GGTAACTACGTTAAAATGGTTCACAACGGTATCGAATATGGCGATATGCAACTCATCTCC GAGTCTTACCTGCTGATGAAAAACATTCTGAACTACAATAACGAGAAACTGAGCGAAGTA TTCAAAAAATGGAACGAAGGTATCCTCAACTCTTATCTGATCGAAATCACCTATAAGATT CTGGGCAAGAAGGACGAACTCACGGACAACCATCTCGTTGACATGATCCTGGACATCGCG GGTGCTAAGGGTACGGGTAAATGGACCATGCTGGAAGCGATCGAGCGTGGTATCCCGTGC CCGACCATGTGTGCGGCGCTGGACGCCCGTAATATCTCCGCGTATAAACAGCTGCGCGTT AAGGCGGACTCTCATTTTTCTAACGACATGAAGGTTAACAAAGTTGAAGGTGAAAATCTC ATTAACTTTGAAGACGACCTCCTGAATGCGCTGTACTGCTGCAAGATCATTTCTTACACC CAGGGTCTGTTCCTGCTCAAACAGGTTTCTGAAGAGATGAAGTGGAATCTCAATCTGGGT GAAATCTCTCGTATTTGGCGTGGTGGCTGCATCATCCGCGCTGTCTTCCTGGATCGTATT ACTAACGCGTACAAGAAGAATGAAAAGCTCGACCTGCTCTTTCTCGACGAGGATTTCGCG CAGGAAATGAAGAACAAACTCCCGAGCCTGCGTAAAGTTGTTCAAGTTGCGACCAAATCT TCTATCCCGATCCCGGCGTTCAGCGCGTCTCTGGCCTACTTTCAGATGGTGACCTCTCAA AACCTCCCACTGAACCTGGTGCAGGCGCAACGTGACTACTTCGGCTCTCACACGTATAAG CGTGTTGACCGTGACGGTGACTTCCATACTATCTGGGAATAACAGTAAAGGTGGATA *GC% Content for gene (codon optimized): 49.2%
source: http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
*took CDS gene sequence from primer design tails protocol and input into Oligo Analyzer with 0.4uM Oligo concentration, 50mM Na+ concentration, 2mM Mg concentration and 0.3mM dNTP concentration.
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) https://drive.google.com/?pli=1&authuser=0&urp=http://www.google.com/accounts/Logout2?hl%3Den_US%26se#folders/0B_Gl3lMyhDsoSmpQWmV3QTB5OTQ
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Forward primer: 5’ TACTTCCAATCCATGACCGGCACTTGTGATAT 3’
*NCBI Gene # or RefSeq#: 5475141
*Protein ID (NP or XP #) or Wolbachia#: PVX_117745 (PlasmoDB)
*Organism (including strain): Plasmodium vivax
Etiologic Risk Group (see link below):
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Plasmodium vivax is a protozoal parasite that is the main source of malaria. P. vivax is one of six species of malaria carriers that infect humans.
Symptoms include fever, chills, headache, muscular aching and weakness, vomiting, cough, diarrhoea and abdominal pain. According to DNDi (Drugs for Neglected Diseases initiative), malaria kills about one child every 30 seconds.
Link to TDR Targets page (if present):
http://tdrtargets.org/targets/view?gene_id=268632
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein:
Gene/Ortholog: mtu1874 (OG4_10600); Phenotype: non-essential; Source study: nmpdr
Gene/Ortholog: mtu1142 (OG4_10600); Phenotype: essential; Source study: nmpdr
Gene/Ortholog: eco1968 (OG4_10600); Phenotype: undefined; Source study: blattner
Gene/Ortholog: eco1968 (OG4_10600); Phenotype: non-essential; Source study: gerdes
Gene/Ortholog: eco1968 (OG4_10600); Phenotype: non-essential; Source study: keio
Gene/Ortholog: eco1968 (OG4_10600); Phenotype: non-essential; Source study: shigen
Gene/Ortholog: cel16317 (OG4_10600); Phenotype: Larval/Adult Lethal/Arrest; Source study: neb
Gene/Ortholog: cel16317 (OG4_10600); Phenotype: Embryonic Lethal/Arrest; Source study: neb
Gene/Ortholog: cel16317 (OG4_10600); Phenotype: Embryonic Lethal/Arrest; Source study: wormbase
Gene/Ortholog: cel16317 (OG4_10600); Phenotype: Larval/Adult Lethal/Arrest; Source study: wormbase
Gene/Ortholog: Tb09.211.3180 (OG4_10600); Phenotype: significant loss of fitness in bloodstream forms (3 days); Source study: alsford
Gene/Ortholog: Tb09.211.3180 (OG4_10600); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford
Gene/Ortholog: Tb09.211.3180 (OG4_10600); Phenotype: significant loss of fitness in procyclic forms; Source study: alsford
Gene/Ortholog: Tb09.211.3180 (OG4_10600); Phenotype: significant loss of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Complex of proteins?: No, but the active site may be in chain A or B
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.8
*EC#: 1.1.1.44
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.44
PDB Site:
http://www.rcsb.org/pdb/explore/explore.do?structureId=2W8Z (not of actual organism, but query cover of 98% and ident of 50% homology structure).
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): continuous spectophotometric rate determination
-- link to Sigma (or other company) page for assay (see Sigma links below)
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/6phosphoglucdehydrog75.pdf.
-- -or link (or citation) to paper that contains assay information
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
http://www.sigmaaldrich.com/catalog/search?interface=All&term=glycylglycine&N=0&mode=match%20partialmax&focus=product&lang=en®ion=US
Glycylglycine:
Quantity:132.12g
Cost:$1,170.00
Supplier: PharmaGrade
Catalog: G0674-100G
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: Homology model: PDB 2W8Z w/ lig
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors:
Expression Information (has it been expressed in bacterial cells): expressed in E. coli bacterial cells
Purification Method:
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MTGTCDIGLIGLAVMGQNLSLNIASNGFTIGVYNRTYERTEDTLKKAKEGNLPIQGYETL EQLINNLKKPRKIILLIKAGPAVDETIKNILKHFEEGDIIIDGGNEWYLNTERRITLCEE HKVEYLAMGVSGGEAGARYGCSFMPGGSKYAYDTIKDILEKCSAKVGTSPCVTYIGPRSS GNYVKMVHNGIEYGDMQLISESYLLMKNILNYNNEKLSEVFKKWNEGILNSYLIEITYKI LGKKDELTDNHLVDMILDIAGAKGTGKWTMLEAIERGIPCPTMCAALDARNISAYKQLRV KADSHFSNDMKVNKVEGENLINFEDDLLNALYCCKIISYTQGLFLLKQVSEEMKWNLNLG EISRIWRGGCIIRAVFLDRITNAYKKNEKLDLLFLDEDFAQEMKNKLPSLRKVVQVATKS SIPIPAFSASLAYFQMVTSQNLPLNLVQAQRDYFGSHTYKRVDRDGDFHTIWE
*length of your protein in Amino Acids: 473 aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 53372.4
Molar Extinction coefficient of your protein at 280 nm wavelength: 66405 assuming all pairs of Cys residues form cystines
Literature:
http://www.sciencedirect.com/science/article/pii/S1047847709002159
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2939770/
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
TACTTCCAATCCATGACCGGCACTTGTGATATCGGTCTCATTGGTCTGGCGGTTATGGGTCAGAACCTGTCC
CTGAACATCGCCTCTAACGGTTTTACCATCGGTGTTTACAACCGTACCTACGAGCGTACG
GAAGATACCCTGAAGAAAGCGAAAGAGGGTAACCTGCCGATCCAAGGTTATGAAACCCTC
GAGCAGCTGATCAACAACCTCAAGAAACCGCGTAAAATCATTCTGCTCATCAAAGCGGGT
CCGGCGGTTGACGAAACCATTAAGAACATCCTGAAGCACTTTGAGGAAGGCGACATCATC
ATCGACGGTGGTAACGAATGGTACCTCAATACCGAACGTCGTATCACTCTGTGCGAAGAG
CACAAAGTAGAATACCTGGCGATGGGTGTTTCTGGTGGCGAAGCAGGTGCGCGCTATGGC
TGTTCTTTCATGCCGGGTGGCTCCAAATATGCGTATGACACGATCAAAGACATCCTCGAG
AAGTGCTCTGCGAAAGTTGGCACGAGCCCTTGCGTAACTTACATCGGTCCTCGTAGCTCT
GGTAACTACGTTAAAATGGTTCACAACGGTATCGAATATGGCGATATGCAACTCATCTCC
GAGTCTTACCTGCTGATGAAAAACATTCTGAACTACAATAACGAGAAACTGAGCGAAGTA
TTCAAAAAATGGAACGAAGGTATCCTCAACTCTTATCTGATCGAAATCACCTATAAGATT
CTGGGCAAGAAGGACGAACTCACGGACAACCATCTCGTTGACATGATCCTGGACATCGCG
GGTGCTAAGGGTACGGGTAAATGGACCATGCTGGAAGCGATCGAGCGTGGTATCCCGTGC
CCGACCATGTGTGCGGCGCTGGACGCCCGTAATATCTCCGCGTATAAACAGCTGCGCGTT
AAGGCGGACTCTCATTTTTCTAACGACATGAAGGTTAACAAAGTTGAAGGTGAAAATCTC
ATTAACTTTGAAGACGACCTCCTGAATGCGCTGTACTGCTGCAAGATCATTTCTTACACC
CAGGGTCTGTTCCTGCTCAAACAGGTTTCTGAAGAGATGAAGTGGAATCTCAATCTGGGT
GAAATCTCTCGTATTTGGCGTGGTGGCTGCATCATCCGCGCTGTCTTCCTGGATCGTATT
ACTAACGCGTACAAGAAGAATGAAAAGCTCGACCTGCTCTTTCTCGACGAGGATTTCGCG
CAGGAAATGAAGAACAAACTCCCGAGCCTGCGTAAAGTTGTTCAAGTTGCGACCAAATCT
TCTATCCCGATCCCGGCGTTCAGCGCGTCTCTGGCCTACTTTCAGATGGTGACCTCTCAA
AACCTCCCACTGAACCTGGTGCAGGCGCAACGTGACTACTTCGGCTCTCACACGTATAAG
CGTGTTGACCGTGACGGTGACTTCCATACTATCTGGGAATAACAGTAAAGGTGGATA
*GC% Content for gene (codon optimized): 49.2%
source: http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
*took CDS gene sequence from primer design tails protocol and input into Oligo Analyzer with 0.4uM Oligo concentration, 50mM Na+ concentration, 2mM Mg concentration and 0.3mM dNTP concentration.
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
https://drive.google.com/?pli=1&authuser=0&urp=http://www.google.com/accounts/Logout2?hl%3Den_US%26se#folders/0B_Gl3lMyhDsoSmpQWmV3QTB5OTQ
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Forward primer:
5’ TACTTCCAATCCATGACCGGCACTTGTGATAT 3’
Reverse primer:
5’ TATCCACCTTTACTGTTATTCCCAGATAGTATGGAAGTC 3’
Products for Enzyme Activity Assay:
N8035-15VL - Beta NADP+
6PG: P6888 - 10mg